The present invention is generally in the area of purification of the naturally produced biological apatite crystals of bone, and of highly purified, calcium-phosphate apatite crystals of bone produced by these methods.
Calcium hydroxyapatites occur naturally as geological deposits and in normal biological tissues, principally bone, cartilage, enamel, dentin, and cementum of vertebrates and in many sites of pathological calcifications such as blood vessels and skin. Synthetic calcium hydroxyapatite is formed in the laboratory either as pure Ca.sub.10 (PO.sub.4).sub.6 (OH).sub.2 or hydroxyapatite that is impure, containing other ions such as carbonate, fluoride, chloride for example, or crystals deficient in calcium or crystals in which calcium is partly or completely replaced by other ions such as barium, strontium and lead. Essentially none of the geological and biological apatites are "pure" hydroxyapatite since they contain a variety of other ions and cations and may have different ratios of calcium to phosphorous than the pure synthetic apatites. In general, the crystals of pure synthetic apatites, geological apatites and many impure synthetically produced apatites are larger and more crystalline than the biological crystals of bone, dentin, cementum and cartilage.
The calcium-phosphate (Ca--P) crystals of the bones of essentially all vertebrates have the basic crystal structure of hydroxyapatite [Ca.sub.10 (PO.sub.4).sub.6 (OH).sub.2 ] as determined by x-ray diffraction. Indeed, the calcium-phosphate (Ca--P) crystals of essentially all of the normally mineralized tissues of vertebrates, including enamel, dentin, cementum, and calcified cartilage, have the same general crystal structure. There are few exceptions, notably the enamel of shark teeth which have fluoride ions substituted for many of the hydroxyl groups.
However, the crystals of Ca-P found in biological tissues such as bone also contain other atoms and ions such as acid phosphate groups (HPO.sub.4.sup.-2), and carbonate ions (CO.sub.3.sup.-2), which do not occur in pure, synthetic hydroxyapatite. There is also good evidence that bone crystals either do not contain hydroxyl groups, or contain only very few such groups (Bonar, et al., "Structural and composition studies on the mineral of newly formed dental enamel: a chemical, x-ray diffraction, and .sup.31 p and proton nuclear magnetic resonance study" J. Bone Min. Res. 6:1167-1176 (1991), and is therefore more appropriately referred to as "apatite" rather than "hydroxyapatite". Moreover, many of the carbonate and phosphate groups in bone crystals are, from the structural and physical chemical points of view, unstable and very reactive, thus providing certain physical chemical and biological functional and chemical features important in the formation and dissolution of the crystals in biological tissues.
Recent important .sup.31 p-nuclear magnetic resonance spectroscopy studies have also demonstrated that the short-range order or environment of the HPO.sub.4.sup.-2 groups in bone crystals are distinctly different than the HPO.sub.4.sup.-2 groups in synthetic apatites and other related calcium-phosphate crystals (Wu, Ph.D. thesis M. I. T., "Solid state NMR study of bone mineral", August, 1992). These differences in chemical, structural, and short range order of the bone crystals compared with pure, synthetic hydroxyapatite also reflect significant differences in their reactivity and hence in their potential function in a biological environment.
The crystals of bone, dentin and cementum are very small, irregularly shaped, very thin plates whose rough average dimensions are approximately 10 to 50 angstroms in thickness, 30 to 150 angstroms in width, and 200 to 600 angstroms in length. This results in their having a very large surface area to present to the extracellular fluids which is critically important for the rapid exchange of ions with the extracellular fluids. This "ion-reservoir" function of the inorganic crystals is very important for a number of critical biological functions.
The vast majority of the Ca-P crystals of bone are located within the collagen fibrils of bone, as reported by Glimcher, M. J., "A basic architectural principle in the organization of mineralized tissues" In: Milhaud, A. G., ed. Proceedings of the Fifth European Symposium on Calcified Tissues, Bordeaux, France, 1968, Lee and Glimcher, "Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone", J. Mol. Biol. 217: 487-501 (1991); and Glimcher M J, "Molecular biology of mineralized tissues with particular reference to bone" Rev. Mod. Physics 31:359-393 (1959). In general, bone contains approximately 35% organic constituents, the major component being collagen fibrils. See, for example, Cohen-Solal, et al., "Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix: the presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen" Biochem. J. 177:81-98 (1979). Due to their intimate physical location and interrelationship with the collagen fibrils, it has not heretofore been possible to separate and isolate the crystals of bone from the collagen fibrils of bone and other organic constituents of the tissue without producing significant changes in the chemistry, structure, degree of crystallinity and size of the crystals, as reported by Sakae, et al., "Changes in bovine dentin mineral with sodium hypochlorite treatment", J. Dental Res. 1229-1234 (1988).
Methods previously used to remove and isolate the calcium-phosphate apatite crystals of bone have not been successful, either because they do not completely separate the crystals from the organic constituents and/or because they alter the chemistry and structure of the crystals. For example, hydrazine treatment of well mineralized bone carried out at temperatures of 50.degree. C. and higher yielded crystals containing significant amounts of organic constituents and induced significant changes in the crystals. Similarly, while substances such as sodium hypochlorite released calcium-phosphate apatite crystals from bone and other tissues, it was used in the form of an aqueous solution. Contact of bone crystals with water for even short periods of time has been shown to significantly alter the crystals by dissolution, reorganization, re-precipitation, and cannot be prevented by adding calcium and phosphate ions to the water based solution. See, for example, Landis, et al., "Electron microscopic observations of bone tissues prepared by ultracryomicrotomy" J. Ultrastruct. Res. 59:185-206 (1977); Landis, et al., "Electron microscopic observations of bone tissue prepared anhydrously in organic solvents" J. Ultrastruct. Res. 59:1-30 (1977); and Landis, et al., "Electron diffraction and electron probe microanalysis of the mineral phase of bone tissue prepared by anhydrous techniques" J. Ultrastruct. Res. 63:188-223 (1978). Furthermore, it has been found that the crystals are not only altered but also contains significant amounts of organic matrix. In a similar fashion, plasma ashing of bone to remove the organic matrix and disperse the crystals has been shown to induce major alterations in the crystal which as in the other methods described above can also contain significant amounts of organic constituents. Such treatment seriously alters the chemistry and structure of the crystals.
The synthetic materials are highly diverse, as reported in the literature. For example, the characterization of four commercial apatites was reported by Pinholt, et al., J. Oral Maxillofac. Surg. 50(8), 859-867 (August 1992); J. Cariofac. Surg. 1(3), 154-160 (July 1990) reports on a protein, biodegradable material; Pinholt, et al., Scand. J. Dent. Res. 99(2), 154-161 (April 1991) reports on the use of a bovine bone material called Bio-Oss.TM.; Friedman, et al., Arch. Otolaryngol. Head Neck Surg. 117(4), 386-389 (April 1991) and Costantino, et al., Arch. Otolaryngol. Head Neck Surg. 117(4), 379-384 (April 1991) report on a hydroxyapatite cement; Roesgen, Unfallchirurgle 16(5), 258-265 (October 1990), reports on the use of calcium phosphate ceramics in combination with atogeneic bone; Ono, et al., Biomaterials 11(4), 265-271 (May 1990) reports on the use of apatite-wollastonite containing glass ceramic granules, hydroxyapatite granules, and alumina granules; Passuti, et al., Clin. Orthop. 248, 169-176 (November 1989) reports on macroporous calcium phosphate ceramic performance; Harada, Shikwa-Gakuho 89(2), 263-297 (1989) reports on the use of a mixture of hydroxyapatite particles and tricalcium phosphate powder for bone implantation; Ohgushi, et al., Acta Orthop.Scand. 60(3), 334-339 (1989) reports on the use of porous calcium phosphate ceramics alone and in combination With bone marrow cells; Pochon, et al., Z-Kinderchir. 41(3), 171-173 (1986) reports on the use of beta-tricalcium phosphate for implantation; and Glowacki, et al., Clin. Plast. Surg. 12(2), 233-241 (1985), reports on the use of demineralized bone implants. No general conclusions can be drawn from these representative reports except that the need for materials which are useful in fixation of implants and in repair or replacement of bone defects remains and that the materials now available do not solve the many problems associated with the treatment of these problems, due to many variables, including the properties of the materials as well as the ease with which they can be manufactured and utilized by the surgeon.
The majority of synthetic hydroxyapatite preparations that have been proposed for use as bone inductors (to induce bone formation) and osteoconductors (by acting as scaffolds to facilitate for the continuous progression of new bone formation) are of synthetic origin and distinct structurally and chemically from the biological calcium, phosphate crystals in bone. All of these apatites are not only chemically and structurally distinct from the apatite crystals of bone, especially in their short range order, size and reactivity, but in some cases, they contain varying amounts of amorphous calcium-phosphate, that is, calcium-phosphate solids which are not crystalline at all. In other instances, the calcium-phosphates made synthetically also contain calcium salts other than apatite crystals such as calcium oxides. To date, it has not been shown how these additional calcium salts are biocompatible or without untoward effects, either biologically or structurally, nor how they affect the bonding strength between the synthetic apatites used to coat the surfaces of artificial joints implanted to bone and the surface of the artificial joint, and between the synthetic apatites and the bone into which the device is implanted.
It is therefore an object of the present invention to provide the biologically, naturally formed crystals of bone a purified apatite that are substantially free of organic material but which also consist predominantly of highly uniform crystals with respect to the chemistry, structure, size, shape and index of crystallinity.
It is a further object of the present invention to provide methods for the further purification of bone apatite crystals that remove essentially all organic material without disrupting the natural crystalline structure of the bone crystals.